Detection of the Heterogeneous O-Glycosylation Profile of MT1-MMP Expressed in Cancer Cells by a Simple MALDI-MS Method

Abstract

BackgroundGlycosylation is an important and universal post-translational modification for many proteins, and regulates protein functions. However, simple and rapid methods to analyze glycans on individual proteins have not been available until recently.Methods/Principal FindingsA new technique to analyze glycopeptides in a highly sensitive manner by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using the liquid matrix 3AQ/CHCA was developed recently and we optimized this technique to analyze a small amount of transmembrane protein separated by SDS-PAGE. We used the MALDI-MS method to evaluate glycosylation status of membrane-type 1 matrix metalloproteinase (MT1-MMP). O-glycosylation of MT1-MMP is reported to modulate its protease activity and thereby to affect cancer cell invasion. MT1-MMP expressed in human fibrosarcoma HT1080 cells was immunoprecipitated and resolved by SDS-PAGE. After in-gel tryptic digestion of the protein, a single droplet of the digest was applied directly to the liquid matrix on a MALDI target plate. Concentration of hydrophilic glycopeptides within the central area occurred due to gradual evaporation of the sample solution, whereas nonglycosylated hydrophobic peptides remained at the periphery. This specific separation and concentration of the glycopeptides enabled comprehensive analysis of the MT1-MMP O-glycosylation.Conclusions/SignificanceWe demonstrate, for the first time, heterogeneous O-glycosylation profile of a protein by a whole protein analysis using MALDI-MS. Since cancer cells are reported to have altered glycosylation of proteins, this easy-to-use method for glycopeptide analysis opens up the possibility to identify specific glycosylation patterns of proteins that can be used as new biomarkers for malignant tumors.

Bilayer Membrane Modulation of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Structure and Proteolytic Activity

“…It is known that the MT1-MMP linker is extensively glycosylated 48 , and glycosylation regulates MT1-MMP substrate targeting 49 . Glycosylation may make the linker more constrained due to steric interactions and/or association with the membrane.…”

Intra-tumor heterogeneity of cancer cells and its implications for cancer treatment

“…Since then, heterogeneous modifications of multiple signaling proteins have been identified, including STAT, ERK, p38, GSK3β, β-catenin, NF-κB, AKT, PTEN, S6 and Src family kinases 28 , 35 , 36 , 37 , 38 . In addition, intra-tumor heterogeneity in O -glycosylation has also been reported 39 .…”

Matrix metalloproteinase collagenolysis in health and disease

“…Gly 272 is critical for the collagenolytic activity of MMP-1, with its role proposed to be the linker-bending motion that allows the HPX domain to present collagen to the CAT domain [99, 100]. MMP-1 and MMP-8 linkers are considerably shorter than the MMP-3 linker, while MT1-MMP linker is very long (33 residues), with significant and heterogeneous O -glycosylation [101]. Thus, linker length per se is not the ultimate criteria for efficient collagenolysis.…”